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Cannabinoid receptors (CBRs) play a key role in various physiological processes, including neurogenesis, synaptic plasticity, immune modulation, cell apoptosis, metabolism regulation, cardiovascular and reproductive systems activity. Since activation of CBRs suppresses hyperexcitation and protects cells from death, their modulation may have therapeutic prospects in the treatment of such pathologies of the nervous system as mental disorders, epilepsy, Parkinson's and Huntington's disease, multiple sclerosis, spinal cord and brain injuries. This paper presents experimental data on the effects of the cannabinoid receptor agonist WIN 55,212-2 on the induced oscillations of intracellular Ca2+ concentration ([Ca2+]i) in two in vitro models of epileptiform activity. To study the neuroprotective properties of WIN 55,212-2, hyperexcitation was induced by the application of a GABA(A) receptor antagonist, bicuculline, or depolarizing doses of ammonium chloride. As experiments have shown, WIN 55,212-2 at a concentration of 100 nM and above significantly suppresses the [Ca2+]i oscillations frequency and reduces the basal [Ca2+]i level. At the same time, the amplitude of calcium oscillations also decreased in the presence of the agonist. WIN 55,212-2 at a concentration of 2 μM suppressed NH4Cl-induced [Ca2+]i oscillations in all neurons but caused a transient biphasic increase in the basal [Ca2+]i level in 20% of astrocytes. Thus, in this work, using various models of hyperexcitation of neuronal networks, we have demonstrated a potential antiepileptic effect of the cannabinoid receptor agonist WIN 55,212-2. [ABSTRACT FROM AUTHOR]

Oscillation in calcium levels in the cytoplasm of individual cells has been observed experimentally to consist of a series of spikes and plateaux of differing amplitudes and inter-peak intervals. On the other hand, mathematical models based on known biochemical reaction kinetic behaviours predict, in the main, limit cycle behaviour. Chaotic solutions do not mimic the observed variability, and so another solution was sought by the introduction of filtered noise into some of the kinetic coefficients. Some of the variability can be predicted from this mechanism, but it is likely that other sources contribute to this as well. [ABSTRACT FROM AUTHOR]

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca2+ levels. In this model, TJs were assumed to be specifically controlled by the Ca2+ concentration levels at the extracellular Ca2+ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca2+ concentration (Ca2+ cell), which might result from changes \ of extracellular Ca2+ concentration (Ca2+ extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca2+-switch experiments were performed in which changes of Ca2+ cell were induced using the Ca2+ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca2+ cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca2+ cell never led to a decline of G, indicating that a rise of Ca2+ cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca2+ cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca2+ cell is without effect on the TJs, since an increase of Ca2+ cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca2+ cell induced by the Ca2+ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca2+ concentration (Ca2+ bl) is increased after TJs were opened by Ca2+ bl removal cannot be ascribed to a rise of Ca2+ cell and might be a consequence of Ca2+ binding to extracellular Ca2+ sites. [ABSTRACT FROM AUTHOR]

Increased lysophosphatidylcholine (LPC) production by the ischemic heart is associated with tissue damage. In vitro, LPC produces an increase in cytosolic [Ca2+], usually followed by cell contracture and lysis. Since ethanol reportedly protect cells during ischemia-reperfusion, we wished to determine whether ethanol could protect heart cells against LPC-induced Ca2+ overload. Newborn rat heart cells in culture were loaded with Fura-2 and [Ca2+]i recorded in individual cells. The presence of 22 or 44 mM ethanol increased the time required for 10 µM L-palmitoyl-LPC to produce maximal Ca2+ accumulation from 8.4±0.4 min (n=47) to 21.1±2.1 min (n=32; P<0.01) and 23.8±1.8 min (n=10; P<0.01) respectively. The onset of the [Ca2+]i increase could be reversed partially by the addition of ethanol (44 or 88 mM). After the addition of 22 mM ethanol, the cells retained the Fura-2 three times longer than under control conditions. Ethanol (88 mM) decreased the critical micelle concentration of LPC, thus decreasing the LPC monomer concentration in this solution. La3+ also protected the cells against LPC but no further protection was afforded by the addition of ethanol. Our results suggest that ethanol concentrations commonly found in the blood of social drinkers protect heart cells against the deleterious effect of LPC. [ABSTRACT FROM AUTHOR]

The intracellular calcium pool of adipose tissue was estimated by studying the kinetics ofCa efflux from the tissue of 9-week old spontaneously hypertensive (SHR) and normotensive Kyoto-Wistar (NKWR) rats subjected to peripheral immunosympathectomy (nerve growth factor antiserum treatment of newborn rats) and in intact (shamtreated) animals of both strains. The development of hypertension in SHR was prevented by immunosympathectomy, however, both groups, SHR-intact and immunosympathectomized rats showed an increase in the intracellular calcium content of adipocytes. These results indicate that the alteration of the membrane control over intracellular calcium distribution in adipose tissue of SHR cannot be attributed to an enhanced activity of the sympathetic nervous system. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum is generally absent from schematic representations of transport phenomena, although it shows a well-organized network in most transport epithelial cells. In order to examine the correlation between this organelle and cellular activity, bladders of Bufo marinus were studied under different experimental conditions and fixed by immersion in glutaraldehyde, followed by OsO impregnation for 3 days. Normal granular and mitochondria-rich cells showed a rich cytoplasmic network of canaliculi, well-impregnated by osmium deposits. Following a 2 to 15-min stimulation (serosal bath) with arginine vasopressin, the V receptor agonist dD-arginine-vasopressin or cyclic AMP (cAMP), the staining of endoplasmic reticulum in granular cells disappeared. After washing out of the hormone or the agonist, impregnation of the endoplasmic reticulum could be observed once again. Arginine vasopressin did not modify the impregnation of endoplasmic reticulum of either mitochondria-rich or basal cells. Our data indicate a correlation between the reactivity of endoplasmic reticulum to osmium, and a cAMP-dependent effect of arginine vasopressin through its V receptors. Incubation of toad bladders carried out with agents interfering with cellular calcium (calcium ionophores, high or low bath calcium) or with calcium release from the endoplasmic reticulum (TMB-8, thapsigargin) suggested that an early step in the cAMP-dependent effect of arginine vasopressin must involve the release of intracellular calcium from the endoplasmic reticulum. However, calcium ATPases in this organelle do not seem to participate in the hormonal effect. The reversible loss of osmium impregnation induced by arginine vasopressin may represent protein changes in the endoplasmic reticulum accompanying a cAMP-dependent calcium release, from the organelle. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the influence of thyroid hormone on tension development and the intracellular calcium transient in mammalian ventricular muscle. A hyperthyroid (H) state was induced in ferrets by subcutaneous injection of l-thyroxine, 0.3 mg/kg daily, for 2-3 weeks. One-half of the age matched control group (C) were injected with vehicle. Aequorin was loaded into the cells of ferret papillary muscles by a chemical procedure. The muscles were stimulated at 0.33 Hz and isometric tension and the calcium transient were simultaneously recorded at 30°C. Peak isometric tension in mN/mm (±SD) was 15.4±7.2 and 16.2±7.9 for C ( n=8) and H ( n=9) respectively. The time to peak tension and time to 80% relaxation from peak of tension were reduced by 22% and 28% respectively in H compared to C. After stimulation, the calcium transient reached a maximum in 56±6 ms in C and in 47±5 ms in H. The time to 80% decay of the peak calcium transient was 95±8 ms and 68±5 ms for C and H respectively. The ratio of the aequorin luminescence at the peak of the calcium transient over the calculated maximum luminescence, L, were compared and they were not different. At 22°C Log ( L/L) was −3.3±0.1 in C ( n=4) and −3.4±0.3 in H ( n=3). These results indicate that the thyroid state influences the time course of the calcium transient and are consistent with the abbreviation in the duration of contraction that is observed in the hyperthyroid state. [ABSTRACT FROM AUTHOR]

The bioluminescent calcium indicator aequorin was successfully loaded into mammalian working myocardium of ferrets by a chemical procedure which makes the cells reversibly hyperpermeable through exposure to Ethylenebis-(oxyethylenenitrilo) tetraacetic acid (EGTA). After undergoing the loading procedure, developed tension at L was 103±26% of the control, which indicates that the muscles regained normal function. The configurations of the aequorin signals (i.e., calcium transients) and their responses to drugs were the same as reported after microinjection of aequorin. The peak of the Ca transient determined by the method of fractional luminescence at 3s intervals of stimulation, 2.5mM [Ca], 22.5°C was 1.1μM; this value is similar to that reported for microinjection. These results indicate that the chemical loading procedure is a useful alternative to microinjection for loading aequorin into mammalian working myocardium. [ABSTRACT FROM AUTHOR]

The bioluminescent calcium indicator aequor in was successfully loaded into vascular smooth muscle cells of Amphiuma tridactylum by either microinjection or a new method which makes the cells reversibly hyperpermeable. Both gave similar results; however, the latter method produced larger signals. Vasoconstrictors produced a sustained contraction and a light (calcium) response consisting of two component: a large transient followed by a smaller, sustained response. Electrical stimulation produced a light transient that was much briefer than the contraction. These results suggest that tension can be maintained in smooth muscle in the presence of lower calcium levels than those present during force development. [ABSTRACT FROM AUTHOR]